The objective of this project is to define the initial, intra-cellular events of steroid hormone action. Synthetic glucocorticoid derivatives, some of which could react to form covalently labeled receptors via affinity labeling, are being used with glucocorticoid-responsive rat hepatoma tissue culture cells to examine: (1) steroid-receptor binding site interactions; (2) the effects of steroid binding on receptor conformation; and (3) the nature of "activation" of receptor-steroid complexes. One these synthetic steroids (dexamethasone 21-mesylate) has been patented as the first irreversible antiglucocorticoid. Using antibodies to rat liver glucocorticoid receptors, we have shown that dexamethasone 21-mesylate covalently labels the receptor in the steroid binding site. A comparison of the steroid mediated events in closely related cell lines has also been used to study steroid hormone action. With this approach, evidence for a new level of complexity in steroid hormone action has been obtained.